Cellular differentiation is associated with transcriptional induction of distinct sets of tissue-specific genes whose expression is required for organ function. Deciphering the mechanisms that control tissue-specific transcription is, thus, critical to understanding cellular differentiation. We have utilized the transthyretin (TTR) DNA regulatory regions as a model in seeking to understand hepatocyte-specific gene transcription. Studies of the TTR promoter suggest that hepatocyte- specific gene regulation relies on combinatorial interaction of multiple DNA binding sites by several distinct families of hepatocyte nuclear factors (HNF). One of these regulatory proteins is the winged helix HNF- 3beta in regulating these genes remains unknown, because homozygous null HNF3beta mice die in utero prior to liver formation. The in vivo role of HNF-3beta in regulating these genes remains unknown, however, because homozygous null HNF-3beta mice die in utero prior to liver formation. In order to examine the regulatory function of HNF-3beta, we inhibited its activity in transgenic mice by increasing hepatocyte expression of the HNF-3beta protein, thus generating a mouse model of liver disease. The mice also display significant reductions in liver glycogen storage and increases in serum bile acid levels. We propose to use them to identify HNF-3beta genes whose altered expression is responsible for this liver phenotype. We recently cloned a Cut-Homeodomain transcription factor, HNF-6, which significantly enhances HNF-3beta and TTR promoter expression. Embryonic expression studies demonstrate that HNF-6 is first expressed in the murine hepatic diverticulum at the onset of liver organogenesis. Although the expression pattern of Hnf-6 and it potential target genes suggest that HNF-6 plays an important role in the induction of liver morphogenesis and hepatocyte differentiation, the exact nature of that role remains unknown. A mouse Hnf6 gene replacement targeting vector will be use to disrupt the murine gene by homologous recombination in embryonic stem (ES) cells, which will then be used to create homozygous null Hnf6 mice. We will use the Hnf6 deficient mice to test the hypothesis that HNF-6 expression is required for liver morphogenesis, hepatocyte differentiation and inn vivo target gene expression. Analysis of the phenotypes in these mice may provide information regarding human birth defects and liver disease resulting from altered expression of differentiating transcription factors.